BRPI0619563A2 - antiviral nucleosides - Google Patents

Abstract

ANTIVIAL NUCLEOSIDS. Compounds having formula i wherein R1 is as defined herein are hepatitis C virus NS5b polymerase inhibitors. Compositions and methods for inhibiting hepatitis replication, and processes for making the compounds of formula I are also disclosed.

Description

ANTIVIAL NUCLEOSIDS

The present invention provides acylated nucleosides which are prodrugs of a hepatitis C virus (HCV) RNA-dependent viral RNA polymerase inhibitor. These compounds when administered orally are easily absorbed. GI tract and efficiently reverted to the parent nucleoside in the blood. These prodrugs are inhibitors of RNA-dependent viral RNA replication and are useful as HCV NS5B polymerase inhibitors, as HCV replication inhibitors, and for the treatment of hepatitis C infection in mammals.

The invention relates to nucleoside prodrugs that are inhibitors of HCV replication. In particular, the invention relates to the use of acylated pyrimidine nucleoside compounds that provide improved drug absorption when the nucleoside is orally administered.

Hepatitis C virus (HCV) is a major health problem and the leading cause of chronic liver disease worldwide (Boyer, N. et al 1 Hepatol. 2000 32: 98-112). HCV-infected patients are at risk of developing liver cirrhosis and subsequent hepatocellular carcinoma, and therefore HCV is the main indication for liver transplantation.

According to the World Health Organization, there are over 200 million people infected worldwide, with at least 3 to 4 million people being infected each year. Once infected, about 20% of people are virus free, but the rest can carry HCV for the rest of their lives. Ten to twelve percent of chronically infected people will eventually develop cirrhosis that destroys the liver or cancer. Viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles or sexually, and vertically from infected mothers or mothers with their offspring. Current treatments for HCV infection, which are restricted to recombinant interferon-α immunotherapy alone or in combination with ribavirin nucleoside analog, are of limited clinical benefit as resistance develops rapidly. There is an urgent need for better therapeutic agents that effectively combat chronic HCV infection.

HCV has been classified as a member of the Flaviviridae viral family which includes the genus flavivirus, pestivirus and hapaceivirus which includes hepatitis C virus (Rice, cM, Flavivirity: The viruses and their replication. In: Fields Virology, Publishers: Fields, BN , Knipe, DM, and Howley, PM, Lippincott-Raven Publishers, Philadelphia, PA, Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a sense-positive single stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5 'untranslated region (RTU), a long open reading frame encoding an approximately 3011 amino acid polyprotein precursor, and a short 3' RTU. The 5 'UTR is the most highly conserved part of the HCV genome and is important for initiation and control of polyprotein translation.

HCV genetic analysis identified six major genotypes that show> 30% divergence in their DNA sequence. Each genotype contains a series of more closely related subtypes that show a 20-25% divergence in nucleotide sequences (Simmonds, P. 2004 1 Gen. Virol. 85: 3173-88). More than 30 subtypes were distinguished. In the US, approximately 70% of infected people have a type Ia and Ib infection. Type Ib is the most prevalent subtype in Asia (X. Forns and J. Bukh7 Clinics in Liver Disease 1999 3: 693-716; J. Bukh et al Semin Liv Liv Dis 1995 15: 41-63). Unfortunately, type 1 infections are more resistant to therapy than type 2 or 3 genotypes (N. N. Zein, Clin. Microbiol. Rev., 2000 13: 223-235).

The genetic organization and polyprotein processing of the nonstructural protein portion of the pestivirus and hepacivirus ORF are very similar. These positive-stranded RNA viruses have a single large open reading frame (ORF) that encodes all viral proteins required for viral replication. These proteins are expressed as a polyprotein that is processed together and after translation by both cellular and virus-encoded proteinases to generate mature viral proteins. Viral proteins responsible for viral genome RNA replication are located approximately at the carboxy terminus. Two-thirds of the ORF are called nonstructural proteins (NS). For both pestiviruses and hepaciviruses, mature nonstructural (NS) proteins, in sequential order from the amino terminus of the non-structural protein coding region to the 0RF carboxy terminus, consist of p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.

The pestivirus and hepacivirus NS proteins share sequence domains that are characteristic of protein specific functions. For example, virus NS3 proteins in both groups have characteristic amino acid sequence motifs of serine proteinases and helicases (Gorbalenya et al., (1988) Nature 333: 22; Bazan and Fletterick (1989) Virology 171: 637- 639; Gorbalenya et al. (1989) Nucleic Acid Res. 17,388-3897). Similarly, pestivirus and hepacivirus NS5B proteins have the characteristic motifs of RNA-directed RNA polymerases (Koonin, Ε. V. and Dolja, VV (1993). Crit. Rev. Biochem. Molec. Biol. 28: 375-430 ).

The actual roles and functions of pestivirus and hepacivirus NS proteins in the life cycle of viruses are directly analogous. In both cases, serine proteinase NS3 is responsible for the entire proteolytic process of polyprotein precursors below their ORF position (Wiskerchen and Collect (1991) Virology 184: 341-350; Bartenschlager et al., (1993) J. Virol 67: 3835-3844; Eckart et al (1993) Biochem Biophys Res. Conm 192: 399-406; Grakoui et al (1993) J. Virol 67: 2832-2843; Grakoui et al. et al. (1993) Proe Natl. Aead Sci USA90: 10583-10587; Hijikata et al. (1993) J. Virol. 67: 4665-4675; Tome et al. (1993) J. Virol. 67: 4017-4026). NS4A protein, in both cases, acts as a cofactor with NS3 serine protease (Bartenschlager et al., (1994) J. Virol. 68: 5045-5055; Failla et al. (1994) J. Virol. 68 : 3753-3760; Xu et al (1997) J. Viral 71:53 12-5322). The NS3 protein of both viruses also functions as a helicase (Kim et al., (1995) Biochem. Biophys. Res. Comm. 215: 160-166; Jin and Peterson (1995) Arch. Biochem. Biophys., 323: 47-53; Warrener and Collett (1995) J. Virol. 69: 1720-1726). Finally, pestivirus and hepacivirus NS5B proteins have predicted RNA-directed RNA polymerase activity (Behrens et al (1996) EMBO. 15: 12-22; Lechmann et al. (1997) J. Virol. 71: Yuan et al (1997) Biochem Biophys Res. Comm 232: 231-235; Hagedorn, PCT WO 97/12033; Zhong et al. (1998) J. Virol 72.9365-9369) .

There are currently a limited number of approved therapies that are currently available for the treatment of HCV infection. New and existing therapeutic approaches for HCV treatment and HCV NS5B polymerase inhibition have been reviewed: R. G. Gish, Sem. Liver. Dis., 1999 19: 5; Di Besceglie, A.M. and Bacon, B.R., Scientific American, October: 1999 80-85; G Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease, Curr. Drug Targ. Infect Dis. 2003 3 (3): 247-253; F. Hoffmann et al, Recent patents on experimental therapy for Hepatitis C Virus infection (1999-2002), Exp. Opin. The R. Patents 2003 13 (11): 1707-1723; F. F. Poordad et al Developments in Hepatitis C therapy during 2000-2002, Exp. Opin. Emerging Drugs 2003 8 (1): 9-25; Μ P. Walker et al, Promising Candidates for the Treatment of Chronic Hepatitis C, Exp. Opin. Investigation Drugs 2003 12 (8): 1269-1280; S.-L. Tan et al, Hepatitis C Therapeutics: Current Status and Emerging Strategies, Nature Rev. Drug Discov. 2002 1: 867-881; R. De Francesco et al Approaching a new era for Hepatitis C Virus therapy: inhibitors of the serine protease NS3-4A and the NS5B RNA-dependent RNA polymerase, Antiviral Res. 2003 58: 1-16; Q. M. Wang et al. Hepatitis C Virus encoded proteins: targets for antiviral therapy, Drugs of the Future 2000 25 (9): 933-8-944; J.A. Wu and Z. Hong, Targeting NS5B-Dependent RNA Polymerase for Anti-HCV Chemotherapy Cur. Drug Targ.-Inf. Dis. 2003 3: 207-219. The reviews cite compounds currently at various stages of the development process. Combination therapy with two or three agents directed at the same or different targets has become standard therapy to prevent or retard the development of resistant strains of a virus, and the compounds disclosed in the above review may be used in compound combination therapy. of the present invention, and such revisions are incorporated herein in their entirety by reference.

<formula> formula see original document page 7 </formula>

Ribavirin (1α; 1 - ((2R, 3R, 4S, 5R) -3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl) -1H- [1,2,4] triazol-3 acid VIRAZ0LE®) is a broad spectrum non-interferon-inducing synthetic antiviral nucleoside analog. Ribavirin has in vitro activity against various DNA and RNA viruses, including Flaviviridae (Gary L. Davis, Gastroenterology 2000 118: S104-S114). On its own, ribavirin reduces serum amino acid transferase levels to normal in 40% of patients, but it does not decrease serum HCV-RNA levels. Ribavirin also exhibits significant toxicity and is known to induce anemia. Ribavirin is an inosine monophosphate dehydrogenase inhibitor. Ribavirin is not approved on HCVi monotherapy but the compound is approved on combination therapy with interferon a-2a and interferon a-2b. Viramidine Ib is a prodrug converted to la in hepatocytes.

Interferons (IFNs) have been available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. Two distinct types of interferon are recognized: type 1 includes several alpha interferons and one interferon β, type 2 includes interferon γ. Interferon type 1 is mainly produced by infected cells and protects neighboring cells from further infection. IFNs inhibit viral replication of various viruses, including HCV, and when used as the sole treatment for hepatitis C infection, IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary. Cessation of therapy results in a relapse rate of 70% and only 10-15% exhibit a sustained virological response with normal serum alanine transferase levels (L.- B. Davis, supra).

A limitation of early IFN therapy was the rapid clearance of blood protein. Chemical derivatization of IFN with polyethylene glycol (PEG) resulted in proteins with substantially improved pharmacokinetic properties. PEGASYS® is a conjugated interferon Δα-2a and a 40 kD branched PEG mono-methoxy and PEG-INTRON® is an α-2b interferon conjugate and a 12 kD PEG mono-methoxy (BA Luxon et al., Clin. Therap, 2002 24 (9): 13631383 (J. Kozlowski and J. Harris J. Control Releasel 2001 72: 217-224).

Interferon alfa-2a and interferon alfa-2b are currently approved as monotherapy for the treatment of HCV.

ROFERON®-A (Roche) is the recombinant form of interferon alfa-2a. PEGASYS® (Roche) is the pegylated (i.e. modified by polyethylene glycol) form of interferon alfa-2a. INTR0N®A (Schering Corporation) is the recombinant form of Interferon alfa-2b, and PEG-INTRON (Schering Corporation) is the pegylated form of interferon alfa-2b.

Other forms of interferon alfa, as well as interferon β, γ, τ and ω, are currently in clinical development for the treatment of HCV. For example, INFERGEN (interferon alfacon-1) by InterMune, OMNIFERON® (natural interferon) by Viragen, ALBUFERON by Human Genome Sciences, REBIF® (interferon β-la) by Ares — Serono, Interferon Omega by BioMedicine, Interferon Alpha Oral by Amarillo Biosciences and interferon γ, interferon τ and interferon γ-Ib by InterMune are under development.

HCV combination therapy with ribavirin and interferon-α is currently the optimal therapy. The combination of ribavirin and PEG-IFN (infra) results in a sustained viral response in 54-56% of patients. SVR covers 80% for HCV type 2 and 3 (Walker, supra). Unfortunately, the combination also produces side effects that pose clinical challenges. Depression, flu-like symptoms and skin reactions are associated with subcutaneous IFN-α, and hemolytic anemia is associated with sustained ribavirin treatment.

Other macromolecular compounds currently in preclinical or clinical development for the treatment of hepatitis C infection virus include: Interleukin-10 by Schering-Plow, IP SO1 by Intemeuron, Merimebodib (VX-497) by Vertexi HEPTAZ YME® by RPI, IDN-30 6556 by Idun Pharma., XTL-002 by XTL., HCV / MFS9 by Chiron, CIVACIR® (Hepatitis C Immune Globulin) by NABI, ZADAXIN® (Thymosin a-1) by SciClone, Thymosin plus interferon pegylated. SciClone, CEPLENE®; an E2 targeted therapeutic vaccine by Innogenetics, an Intercell therapeutic vaccine, an Epimmune / Genencor therapeutic vaccine, a Merix therapeutic vaccine, a therapeutic vaccine, ChronVacC, a Tripep.

Other macromolecular approaches include HCV RNA-directed ribozymes. Ribozymes are naturally occurring short molecules with endonuclease activity.

An alternative approach is the use of antisense oligonucleotides that bind to RNA and stimulate RNaseH-mediated cleavage.

Numerous potential molecular targets for drug development as anti-HCV therapies have now been identified, including, without limitation, NS2-NS3 autoprotease, N3 protease, N3 helicase and NS5B polymerase. RNA-dependent RNA polymerase is absolutely essential for replication of the positive sense single-stranded RNA genome, and this enzyme has sparked significant interest among medical chemists.

Nucleoside NS5B polymerase inhibitors may act as an unnatural substrate that results in chain termination or as a competitive inhibitor that competes with nucleotide binding to the polymerase. To function as a chain terminator, the nucleoside analog must be absorbed by the cell and converted in vivo to a triphosphate to compete for the polymerase nucleotide binding site. This triphosphate conversion is commonly mediated by cellular kinases that convey additional structural needs in a potential nucleoside polymerase inhibitor. Unfortunately, this limits the direct evaluation of nucleosides as HCV replication inhibitors for cell-based assays capable of in situ phosphorylation.

<formula> formula see original document page 11 </formula>

B = adenine, thymidine, uracil, cytidine, guanine and hypoxanthine

In WO 01 90121, published November 29, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify the anti-HCV polymerase activity of 1'-alkyl- and 2'-alkyl nucleosides of formulas 2 and 3. In WO 01/92282, published December 6, 2001, J.-P. Sommadossi and P. Lacolla disclose and exemplify the treatment of Flaviviruses and Pestiviruses with I1-alkyl- and 2'-alkyl nucleosides of formulas 2 and 3. In WO 03/026675, published April 3, 2003, G. Gosselin discloses 41-alkyl nucleosides for the treatment of Flavivirus and Pestivirus.

In W02004003000, published January 8, 2,004, 30 J.-P. Sommadossi et al. disclose 2 'and 3' prodrugs of 1 ', 2', 3 'and 4'-substituted β-D and β-L nucleosides. In WO 2004/002422, published January 8, 2004, 2'-C-methyl-3'-0-valine ribofuransyl cytidine ester for the treatment of Flaviviridae infections. Idenix reported clinical trials for a related compound NM283 believed to be the valid ester of the cytidine analog 2 (B = cytosine). In WO 2004/002999, published January 8, 2004, J. -P. Sommadossi et al disclose a series of 21 or 3 '1', 2 ', 3', or 4 'branched nucleoside prodrugs for the treatment of flavivirus infections including HCV infections.

In W02 004/04 6331, published June 3, 2004, J. P. Sommadossi et al. disclose 21-branched nucleosides and Flaviviridae mutation. In W003 / 026589, published April 3, 2003, G. Gosselin et al. disclose methods of treating hepatitis C virus using 41-modified nucleosides. In W02005009418, published February 3, 2005, R. Storer et al. disclose nucleoside purine analogs for the treatment of diseases caused by Flaviviridae including HCV.

Other patent applications disclose the use of certain nucleoside analogues to treat hepatitis C virus infection. In WO 01/32153, published May 10, 2001, R. Storer discloses nucleoside derivatives for the treatment of viral diseases. In WO 01/60315, published August 23, 2001, H. Ismaili et al. disclose methods of treating or preventing Flavivirus infections with nucleoside compounds. In WO 02/18404, published March 67, 2002, R. Devos et al. disclose 4'-substituted nucleotides for the treatment of HCV viruses. In WO 01/79246, published October 25, 2001, K. A. Watanabe discloses 2 'or 31-hydroxymethyl nucleoside compounds for the treatment of viral diseases. In WO 02/32920, published April 25, 2002, and in WO 02/48 165, published June 20, 2002, L. Stuyver et al. disclose nucleoside compounds for the treatment of viral diseases.

<formula> formula see original document page 13 </formula>

In WO 03/105770, published December 24, 2003, B. Bhat et al. disclose a series of carbocyclic nucleoside derivatives that are useful for treating HCV infections. In WO 2004/007512, published January 22, 2003, B. Bhat et al. disclose nucleoside compounds that inhibit RNA-dependent viral RNA polymerase. The nucleosides disclosed in this publication are primarily substituted 2'-methyl-2'-hydroxy nucleosides. In WO 2002/057425, published July 25, 2002, S. S. Carroll et al. disclose nucleoside derivatives that are RNA-dependent viral polymerase inhibitors and methods of treating HCV infection. In WO 02/057287, published July 25, 2002, S. S. Carroll et al. disclose related derivatives of 2α-methyl and 2β-methylribose wherein the base is an optionally substituted 7H-pyrrol [2,3-d] pyrimidine 6 radical. The same application discloses an example of a 3 P-methyl nucleoside. S. S. Carroll et al (J. Biol. Chem. 2003 278 (14): 11979-11984) disclose inhibition of HCV polymerase by 2'-O-methylcytidine (6a). In WO 2004/009020, published January 29, 2004, D. B. Olsen et al. disclose a series of thionucleoside derivatives as RNA dependent RNA polymerase inhibitors. PCT Publication No. WO 99/43691 for "Emory

University ", entitled" 21 - Fluoronucleosides ", discloses the use of certain 21-fluornucleosides to treat HCV. US Patent No. 6.34 8,587 to Emory University", entitled "21 - Fluoronucleosides", discloses a family of 2'-fluornucleosides useful for treating HCV. the treatment of hepatitis B, HCV, HIV and abnormal cell proliferation. Both configurations of the 2'-fluorine substituent are revealed.

Eldrup et al (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.)) Have described modified for HCV inhibition.

Bhat et al (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.); Ρ A75) describe the synthesis and pharmacokinetic properties of nucleoside analogues as possible inhibitors of RNA replication. of HCV. The authors report that 21-modified nucleosides demonstrated potent inhibitory activity in cell-based replicon assays.

Olsen et al (Oral Session V, Hepatitis C Virus, Flaviviridae; 16th International Conference 30 on Antiviral Research (Apr. 27, 2003, Savannah, Ga.) Ρ A76) also described the effects of 21-modified nucleosides on HCV RNA replication. . Allosteric non-nucleoside HIV reverse transcriptase inhibitors have proven to be effective therapies alone and in combination with nucleoside inhibitors and protease inhibitors. Several classes of HCV NS5B non-nucleoside inhibitors have been described and are currently in various stages of development including: benzimidazole (H. Hashimoto et al. WO 01/47833, H. Hashimoto et al. WO 03/000254, PL Beaulieu et al WO 03/020240 A2; PL Beaulieu et al US 6,448,281 BI; PL Beaulieu et al WO 03/007945 Al); indoles (P.L. Beaulieu et al. WO 03/0010141 A2); benzothiadiazines, for example, 7 (D. Dhanak et al. WO 01/85172 Al; D. Dhanak et al. WO 03/037262 A2; KJ Duffy et al. W003 / 099801 Al, D.Chai et al. WO 2004052312, D.Chai et al W02004052313, D.Chai et al WOO2 / 098424, JK Pratt et al WO 2004/041818 Al; JK Pratt et al WO 2004/087577 Al), thiophenes, for example 8 (CK Chan et al WO 02/100851);

<formula> formula see original document page 15 </formula>

benzothiophenes (D.C. Young and T.R. Bailey WO 00/18231); β-ketopyruvates (S. Attamura et al US 6,492,423 BI, A. Attamura et al WO 00/06529); pyrimidines (C. Gardelli et al WO 02/06246 A1); pyrimidinediones (T. R. Bailey and D. C. Young WO 00/13708); triazines (K.-H. Chung et al. WO 02/079187 A1); rhodamine derivatives (T. R. Bailey and D. C. Young WO 00/10573, J. C. Jean et al WO 01/77091 A2); 2,4-dioxopyran (R. A. Love et al. EP 256628 A2); phenylalanine derivatives (M. Wang et al. J Biol. Chem. 2003 278: 2489-2495).

NS3 protease has emerged as a major target for the discovery of new anti-HCV therapy. In WO 98/22496, published May 28, 1998, M. R. Attwood et al. disclosed protease mechanism-based active site inhibitors (MR Attwood et al., Antiviral Chemistry and Chemotherapy 1999 10: 259-273; MR Attwood et al., Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474). In WO98 / 17679, published April 30, 1998, R. D. Tung et al. disclosed mechanism-based peptide inhibitors in NS3 protease.

In W099 / 07734, published February 18, 1999, and in WOO0 / 09543, published August 9, 1999, M Llinas-Brunet et al. disclose protease peptide inhibitors. In WOO0 / 59929, published 12 October 2000, Y. S. Tsantrizos et al. disclose macrocyclic tripeptides that are potent inhibitors of HCV NS3 protease. A number of related Boehringer-Ingleheim patents disclose related protease inhibitors and lead to the identification of BILN 2061 tripeptide derivative (M. Llinas-Brunet et al. Biorg. Med. Chem. Lett. 2000 10 (20): 2267-70 J. Med Chem. 2004 47 (26): 6584-94; J. Med. Chem. 2004 47 (7): 1605-1608; Angew. Chem. Int. Ed. Eng. 2003 42 (12): 1356- 60).

Other tripeptide inhibitors identified by Bristol-Myers Squibb were disclosed, among others, in W003 / 099274, published December 4, 2003, in W02 004/032827, published April 22, 2004, in WOO 3/05334 9, published July 3, 2003, W02 005/04 6712, published May 26, 2005 and W02005 / 051410, published June 9, 2005. In W02004 / 072234, published August 26, 2004, and In WO2004 / 093798, published November 4, 2004, additional tripeptide protease inhibitors were disclosed by Enanta Pharmaceuticals. In W02005 / 037214, published April 28, 2005, L. M. Blatt et al. further disclose other tripeptide derivatives that inhibit HCV NS3 protease. In W02005 / 030796, published April 7, 2005, S. Venkatraman et al. disclose macrocyclic HCV serine protease NS3 inhibitors. In WO 2005/058821, published June 30, 2005, F. Velazquez et al. disclose HCV NS3 / NS4a serine protease inhibitors. In W002 / 48172, published June 20, 2002, Z. Zhu discloses diaryl peptides as NS3 protease inhibitors. In W002 / 08187 and W002 / 08256, both published January 31, 2002, A. Saksena et al. disclose HCV NS3 protease peptide inhibitors. In WOO 2/08251, published January 31, 2002, M. Lim-Wilby et al. disclose NS3 protease peptide inhibitors. In US 6,004,933, issued December 21, 1999, L. W. Spruce et al. disclose heterocyclic peptide derivatives that inhibit cysteine proteases including HCV endopeptidase.

Non-substrate-based NS3 protease inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al., BBRC1 238: 643-647,1997; Sudo K. et al., Antiviral Chemistry and Chemotherapy 9: 186,1998), including RD 3-408 and RD 3-4078, the former substituted on the 14-carbon chain amide and the latter processing a para-phenoxyphenyl group, are also being investigated.

SCH 68631, a phenanthrenquinone, is an HCV protease inhibitor (Chu M. et al., Tetrahedron Letters 37: 7229-7232,1996). In another example by the same authors, SCH 351633, isolated from the fungus Penicillixmi griscofuluum, was identified as a protease inhibitor (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9: 1949-1952). Nanomolar potency against HCV NS3 protease enzyme was achieved by design of selective inhibitors based on eglin c macromolecule. Eglin c, isolated from leech, is a potent inhibitor of various serine proteases such as S. griseus Ae B proteases, α-chymotrypsin, chimase and subtilisin. Qasim M. A. et al., Biochemistry 36: 1598-1607,1997.

Thiazolidine derivatives showing relevant inhibition in a reverse phase HPLC assay with a NS3 / 4A fusion protein and NS5A / 5B substrate (Sudo, K. et al., Antiviral Research 32: 9-18, 1996), especially the compound RD-1-6250, which has a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and RD4 6193. Thiazolidines and benzanilides have been identified by N. Kakiuchi et al. in FEBS Letters 421: 217-220; and N. Takeshita et al., Anal. Biochemistry 247: 242-246,1997.

Imidazolidinones as HCV NS 3 serine protease inhibitors are disclosed in WO 02/08198 to Schering Corporation, published January 31, 2002, and WO 02/48157 to Bristol Myers Squibb, published June 20, 2002. In W002 / 48116, published June 20, 2002, P. Glunz et al. disclose NS3 protease pyrimidinone inhibitors.

Other enzymatic targets for anti-HCV include the IRES site of HCV (Internal Ribosomal Entry Site) and HCV helicase. IRES inhibitors have been reported by Immusol, Rigel Pharmaceuticals (R803) and Anadys (ANA 245 and ANA 246). Vertex revealed an HCV helicase inhibitor.

Combination therapy that can suppress resistant mutant strains has become a well-established approach to anti-viral chemotherapy. The nucleoside inhibitors disclosed herein may be combined with other HCV nucleoside polymerase inhibitors, non-nucleoside HCV polymerase inhibitors, and HCV protease inhibitors. Like other HCV drug classes, for example, viral entry inhibitors, helicase inhibitors, IRES inhibitors, ribozymes, and antisense oligonucleotides arise and are developed, they will also be excellent candidates for combination therapy. Interferon derivatives have already been successfully combined with ribavirin and interferons, and chemically modified interferons will be useful in combination with the nucleosides disclosed herein.

Nucleoside derivatives are often potent antivirals (eg, HIV, HCV, Herpes Simplex, CMV) and anti-cancer chemotherapeutic agents. Unfortunately, its practical utility is often limited by two factors. First, poor pharmacokinetic properties often limit intestinal nucleoside absorption and intracellular concentration of nucleoside derivatives, and second, suboptimal physical properties restrict the formulation options that can be employed to improve active ingredient release.

Albert introduced the term prodrug to describe a compound that is devoid of intrinsic biological activity but is capable of metabolic transformation to the active drug substance (A. Albert, Selecium Toxicity, Chapman and Hall, London, 1951). Prodrugs have recently been reviewed (P. Ettmayer et al., J. Med Chem. 2004 47 (10): 2393-2404; K. Beaumont et al., Curr. Drug Metab. 2003 4: 461-485; H. Bundgaard, Design of Prodrugs: Bioreversible derivatives for various functional groups and chemical entities in Design of Prodrugs, H. Bundgaard (ed) Elsevier Science Publishers, Amersterdam 1985; GM Pauletti et al Adv. Drug Deliv. Rev. 1997 27: 235- 256; RJ Jones and N. Bischofberger, Antiviral Res. 1995 27; 1-15 and CR Wagner et al., Med. Res. Rev. 2000 20: 417-45). Although metabolic transformation can be catalyzed by specific enzymes, often hydrolases, the active compound can also be regenerated by non-specific chemical processes.

Pharmaceutically acceptable prodrugs refer to a compound that is metabolised, for example, hydrolyzed or oxidized, in the host to form the compound of the present invention. Bioconversion should prevent the formation of fragments with toxicological hazards. Typical examples of prodrugs include compounds that have biologically unstable protecting groups attached to a functional portion of the active compound. Alkylation, acylation or other lipophilic modification of the hydroxy group in the sugar moiety have been used in the design of pro-nucleotides. Such pro-nucleotides may be hydrolyzed or dealkylated in vivo to generate the active compound.

Factors limiting oral bioavailability are often absorption from the gastrointestinal tract and first pass excretion through the intestinal wall and the liver. Optimization of transcellular absorption through the GI tract requires a D (7.4) greater than zero. Optimization of the distribution coefficient, however, does not guarantee success. The prodrug may have to avoid active efflux transporters in the enterocyte. Intracellular metabolism in the enterocyte may result in passive transport or active transport of the metabolite by efflux pumps back to the intestinal lumen. The prodrug must also resist unwanted biotransformations in the blood before reaching target cells or receptors.

Although supposed prodrugs can sometimes be rationally designed based on the chemical functionality present in the molecule, chemical modification of an active compound produces an entirely new molecular entity that can exhibit undesirable physical, chemical and biological properties missing from the parent compound. Regulatory needs for metabolite identification can present multiple-way challenges that lead to a plurality of metabolites. Therefore, identification of prodrugs remains a challenging and uncertain exercise. In addition, evaluating the pharmacokinetic properties of potential prodrugs is a challenging and costly endeavor. Pharmacokinetic results from animal models may be difficult to extrapolate to humans.

The object of the present invention is to provide novel compounds, methods and compositions for the treatment of a host infected with hepatitis C virus.

The present invention is directed to novel 4-amino-1 - ((2R, 3R, 4R, 5R) -3-fluoro-4-hydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-di-acyl derivatives). 2-yl) -1H-pyrimidin-2-one (also referred to as (2R) 2'-deoxy-2-methyl-2-fluorocytidine). Compounds of the present invention have a structure according to formula I.

<formula> formula see original document page 22 </formula>

on what:

R1 is selected from the group consisting of C2-5 branched or unbranched alkyl, C2-5 branched or unbranched alkenyl, C2-5 branched or unbranched alkynyl, C2-5 lower haloalkyl, C3-6 cycloalkyl, and C2-4 alkoxy; and hydrates, solvates and acid addition salts thereof.

Compounds of the present invention are useful for treating an HCV-mediated disorder.

The invention also comprises methods of treating HCV with compounds of the present invention and pharmaceutical compositions containing said compounds.

In one embodiment of the present invention there is provided a compound according to formula I wherein R 1 is as defined above.

In another embodiment of the present invention there is provided a compound according to formula I wherein R 1 is ethyl, n-propyl, iso-propyl, n-butyl or iso-butyl. In yet another embodiment of the present invention there is provided a compound according to formula I wherein R 1 is ethyl or isopropyl.

In still another embodiment of the present invention there is provided a compound according to formula I wherein R 1 is isopropyl and the compound is a hydrochloride or sulfate salt.

In a further embodiment of the present invention there is provided a compound according to formula I wherein R 1 is isopropyl and the compound is a hydrochloride salt.

In yet another embodiment of the present invention there is provided a compound according to formula I wherein R 1 is ethoxy, n-propoxy or iso-propoxy.

In a still further embodiment of the invention, there is provided a compound according to formula I for use in therapy, particularly for use in the therapy of an HCV virus mediated disease.

In yet another embodiment of the invention, the use of a compound according to formula I for the preparation of a medicament for the treatment of an HCV virus mediated disease is provided.

A compound according to formula I may be particularly used for the preparation of a medicament for administration to a patient in need thereof at a therapeutically effective dose, preferably at a dose of 0.1 to 10 g per day, more preferably. at a dose of 0.5 to 7 g per day, or even more preferably at a dose of 1.0 g to 6.0 g per day.

A compound according to formula I may also be used for the preparation of a medicament which may also comprise a therapeutically effective amount of at least one immune system modulator such as an interferon, a chemically derived interferon, an interleukin, a necrosis factor. or a colony stimulating factor, and / or at least one antiviral agent that inhibits HCV replication, such as HCV protease inhibitor, another HCV nucleoside polymerase inhibitor, a non-nucleoside HCV polymerase inhibitor, HCV helicase inhibitor, an HCV primase inhibitor or an HCV fusion inhibitor.

In still another embodiment of the present invention, there is provided a method for treating an HCV virus mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as defined above. .

In still another embodiment of the present invention, there is provided a method for treating an HCV virus mediated disease comprising administering to a patient in need thereof a dose of 0.1 to 10 g per day of a compound according to the invention. of formula I as defined above.

In yet another embodiment, the dose is between 0.5 and 7 g per day, and in an additional embodiment the dose is between 1.0 and 6.0 g per day.

In another embodiment of the present invention, there is provided a method for treating an HCV virus mediated disease comprising co-administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as above. defined and a therapeutically effective amount of at least one immune system modulator and / or at least one antiviral agent that inhibits HCV replication. In another embodiment of the present invention, there is provided a method for treating an HCV-mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as defined above, and a therapeutically effective amount of at least one immune system modulator whose immune system modulator is an interferon, interleukin, tumor necrosis factor or colony stimulating factor.

In another embodiment of the present invention, there is provided a method for treating an HCV-mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as defined above, and a therapeutically effective amount of at least one immune system modulator whose immune system modulator is a interferon or a chemically derived interferon.

In another embodiment of the present invention, there is provided a method for treating an HCV-mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as defined above, and a therapeutically effective amount of at least one other antiviral compound.

In another embodiment of the present invention, there is provided a method for treating an HCV-mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to formula I as defined above, and a therapeutically effective amount of at least one other antiviral compound whose compound is an HCV protease inhibitor, another HCV nucleoside protease inhibitor, a non-nucleoside HCV protease inhibitor, a HCV helicase inhibitor, a primase inhibitor HCV, or an HCV fusion inhibitor.

In another embodiment of the present invention there is provided a pharmaceutical composition comprising a compound according to formula I as defined above mixed with at least one pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment of the present invention, there is provided a process for the preparation of a compound according to formula I as defined above, which process comprises steps (i) - (v) listed in claim 15 and exemplified in the examples. The process comprises treating I in a basic aqueous organic medium which may be homogeneous or biphasic with an acylating agent as defined herein in the presence of DMAP and sufficient base to maintain the solution at a pH of at least about 7.5. The present process allows acylation without concomitant reaction of the heterocyclic base.

The phrase "one" or "one" entity as used herein refers to one or more of those entities; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "one" (or "one"), "one or more", and "at least one" may be used interchangeably in this specification.

The terms "optional" and "optionally" as used herein mean that a subsequently described event or circumstance may, but need not, occur, and that the description includes cases in which the event or circumstance occurs and cases in which it does not occur. For example, "optional bonding" means that the bonding may or may not be present, and that the description includes single, double or triple bonds.

The term "independently" is used in this specification to indicate that a variable is applied in any case without regard to the presence or absence of a variable having that same definition or a different definition in the same compound. Therefore, in a compound where R appears twice and is defined as "independently carbon or nitrogen", both R may be carbon and the other nitrogen.

The term "alkenyl" as used herein denotes an unsubstituted hydrocarbon chain radical having from 2 to 10 carbon atoms having one or two olefin double bonds [preferably one olefin double bond]. "C 2-10 alkenyl" as used herein refers to an alkenyl composed of 2 to 10 carbons. Examples are vinyl, 1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl).

The term "alkyl" as used herein denotes a saturated branched or unbranched, monovalent hydrocarbon residue containing 1 to 10 carbon atoms.

The term "lower alkyl" denotes a monovalent, straight chain or branched hydrocarbon residue containing 1 to 6 carbon atoms. "C1-10 alkyl" as used herein refers to an alkyl composed of 1 to 10 carbons.

Examples of alkyl groups include, without limitation, lower alkyl, methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl groups. The term (ar) alkyl or (heteroaryl) alkyl indicates that the alkyl group is optionally substituted by an aryl or heteroaryl group respectively.

The term "alkynyl" as used herein denotes a branched or unbranched hydrocarbon chain radical having from 2 to 10 carbon atoms, preferably 2 to 5 carbon atoms, and having a triple bond. "C 2-10 alkynyl" as used herein refers to an alkynyl composed of 2 to 10 carbons. Examples are ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl or 3-butynyl.

The term "cycloalkyl" as used herein denotes a saturated carbocyclic ring containing 3 to 8 carbon atoms, ie cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. "C 3-7 cycloalkyl" as used herein refers to a cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring.

The term "haloalkyl" as used herein denotes a branched or unbranched chain alkyl group as defined above wherein 1, 2, 3 or more hydrogen atoms are substituted by a halogen. "C1-3 haloalkyl" as used herein refers to a haloalkyl composed of 1 to 3 carbons and 1-8 halogen substituents. Examples are 1-fluormethyl, 1-chloromethyl, 1-bromomethyl, 1-iodomethyl, trifluoromethyl, trichloromethyl, tribromomethyl, triiodomethyl, 1-fluoroethyl, 1-chloroethyl, 1-bromoethyl, 1-iodoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-iodoethyl, 2,2-dichloroethyl, 3-bromopropyl or 2,2,2-trifluoroethyl.

The term "alkoxy" as used herein means an -O-alkyl group, where alkyl is as defined above. Examples are methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy. "Lower alkoxy" as used herein denotes an alkoxy group having a "lower alkyl" group as previously defined. "C1-10 alkoxy" refers to an -O- alkyl wherein alkyl is C1-10.

The term "di-acyl" derivative as used herein refers to a nucleoside derivative as described herein wherein the 3 'and 5'-hydroxy are from an ester -OC (= O) R 1 and OC (= 0 ) R2 wherein R1 and R2 are as defined in claim 1.

The term "acylating agent" as used herein refers to an anhydride, acid halide, chlorocarbonylalkoxide (e.g. ethyl chloroformate) or an activated derivative of an N-protected alpha amino acid. The term "anhydride" as used herein refers to compounds of general structure R 1C (O) -O-C (O) R 1 wherein R 1 is as defined in claim 1. The term "acid halide" as used herein refers to to compounds of general structure R 1C (O) X wherein X is a halogen. The term "acyl imidazole" refers to a compound of general structure R 1 C (O) X wherein X is N-imidazolyl. The term "activated derivative" of a compound as used herein refers to a transient reactive form of the parent compound that renders the active compound in a desired chemical reaction, wherein the parent compound is only moderately reactive or nonreactive. Activation is accomplished by formation of a derivative or chemical grouping in the molecule with a higher free energy content than that of the parent compound, which makes the activated form more susceptible to react with another reagent. In the context of the present invention activation of the carboxy group of particular importance and corresponding activating agents or groups which activate the carboxy group are described in more detail below.

Of particular interest to the present invention are carboxylic acid anhydrides and carboxylic acid chlorides.

The phrase "heterogeneous aqueous solvent mixture" as used herein refers to a mixture of water and an organic cosolvent that produces a two-phase or heterogeneous mixture.

Such a heterogeneous aqueous solvent mixture may result from a co-solvent with limited aqueous solubility or the ionic strength of the aqueous component may be adjusted to limit the solubility of the co-solvent in the aqueous phase.

The term "alkali metal hydroxide" refers to a compound of formula MOH wherein M is lithium, sodium, potassium or cesium, "alkali metal bicarbonate" refers to an MHCO3 group where M is sodium or potassium and "alkali metal carbonate" refers to a group M2CO3 wherein M is sodium or potassium. One skilled in the art will appreciate that other bases may be used to maintain the desired pH range and other bases are within the scope of the invention.

Abbreviations used in this application include: acetyl (Ac), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), atmospheres (Atm), high pressure liquid chromatography (HPLC), methyl (Me), tert-butoxycarbonyl ( Boc), acetonitrile (MeCN), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), benzyl (Bn), butyl (Bu), methanol ( MeOH), benzyloxycarbonyl (cbz or Ζ), melting point (mp), carbonyl diimidazole (CDI), MeSO2- (mesyl or Ms), 1,4-diazabicyclo [2.2.2] octane (DABCO), mass spectrum ( ms), methyl t-butyl ether (MTBE), 1,5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), N-Methylmorpholine (NMM), N-Methylpyrrolidone (NMP), 1,2-Dichloroethane (DCE), Ν, N'-Dicyclohexylcarbodiimide (DCC), Pyridinium Dichromate (PDC), Dichloromethane (DCM), Propyl (Pr), pounds per square inch (psi), diisopropylethylamine (DIPEA, Hunig's Base), pyridine (pyr), room temperature, rt or RT, N, N-dimet yl acetamide (DMA), tert-butyldimethylsilyl or t-BuMe2 Si (TBDMS), 4-N, N-dimethylaminopyridine (DMAP), triethylamine (Et3N or TEA), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) trifluoroacetic acid (TFA), thin layer chromatography (TLC), ethyl acetate (EtOAc), tetrahydrofuran (THF), diethyl ether (Et20), trimethylsilyl or Me3 Si (TMS), ethyl (Et), p- toluenesulfonic (TsOH or pTsOH), 4-Me-C6H4SO2- or tosyl (Ts), isopropyl (i-Pr), N-urethane-N-carboxyanhydride (UNCA), ethanol (EtOH). Conventional nomenclature including the prefixes normal (η), iso (i-), secondary (sec-), tertiary (terc-) and neo have the usual meaning when used with an alkyl moiety. (J. Rigaudy and D. P. Klesney, Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.).

Examples of representative compounds encompassed by the present invention and within the scope of the invention are provided in Table I. These following examples and preparations are provided to enable those skilled in the art to more clearly understand and practice the present invention, they should not be considered. as limiting the scope of the invention, but only as being illustrative and representative thereof.

In general, the nomenclature used in this application is based on AUTONOMTM v.4.0, a Beilstein Institute computer system for the generation of IUPAC systematic nomenclature. If there is a discrepancy between a structure shown and a name given to that structure, the structure shown should be given more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated, for example, in bold or dashed lines, the structure or portion of the structure should be interpreted as encompassing all stereoisomers thereof. The enumeration system for these ring systems is as follows:

Compounds and Preparation

The (2R) -2'-deoxy-21-fluorine-21-C-methylcytidine HCV HCV polymerase inhibitory activity has been disclosed (JL Clark et al J. Med. Chem. 2005 48 (17): 5504-8; J. Clark US Publication No. 2005/0009737 and both publications are incorporated herein in their entirety by reference). In clinical practice, it would be desirable to initially administer high doses of 1-6 to rapidly inhibit HCV polymerase and thereby decrease viral levels under conditions that minimize the opportunity for the virus to mutate and to select resistant strains. Sufficiently high levels may be difficult to achieve with the parent nucleoside. Prodrugs provide a strategy for improving the pharmacokinetic and physical properties of a compound and thereby optimizing bioavailability. U.S. Publication 2005/0009737 suggests general approaches to 2'-deoxy-2'-fluoro-21-methyl nucleoside nucleoside prodrugs. Although prodrug candidates are deceptively simple to design, identifying compounds with the appropriate physicochemical and pharmacodynamic properties, in vivo transformations, and safety profile is a complex multi-disciplinary task that requires significant experimentation. Obstacles to the identification of oral release prodrugs include maintaining sufficient aqueous solubility, lipophilia and chemical stability while allowing rapid and efficient release of the active portion after administration. Additionally, significant non-esterase metabolism and mediated clearance of prodrug transporter should be minimized (K. Beaumont et al Curr. Drug Metab. 2003 4 (6): 461-485). Also inhibition or induction of cytochrome P450 enzymes may produce adverse drug-drug interactions that are undesirable. Lower alkyl diesters of 1-6 shown in Table 1 significantly improve the bioavailability of 1-6.

The pharmacokinetic behavior of potential prodrugs was evaluated in rat and monkey to try to minimize intra-species variability and genetic polymorphisms that may result in intra-species artifacts. To the extent that enzymatic transformations are responsible for hydrolysis of ester bonds, prodrug-specific affinity may depend on the specific esterase and / or peptidase structure that may catalyze the transformation. Rat esterase activity was frequently shown to be significantly higher than that of man (J. A. Fix et al Pharm. Res. 1990 7 (4): 384-387;

W. Li et al Antimicrob. Agents Chemother 1998 42 (3): 647-653). Another potentially important parameter was the bioconversion of the cytidine base to uridine by a deaminase. Although cytidine and uridine triphosphates are polymerase inhibitors, in vivo uridine phosphorylation is inefficient compared to cytidine. Thus, increased uridine levels are considered undesirable. The lack of efficacy exhibited by the uridine derivative in the HCV replicon (J. Clark et al J. Med. Chem., Supra) has been reported.

Table 1

<table> table see original document page 34 </column> </row> <table> <table> table see original document page 35 </column> </row> <table>

1. Cmax is the peak concentration of 1-7 in the blood.

2. AUC (cyt) is the area. under the curve, for cytidine nucleoside

AUC (urd) is the area under the uridine nucleoside curve

3. EC90 in replicon eg 5.40 ± 2.6 μΜ (J. Clark et al J. Med. Chem., Supra)

Surprisingly, it has now been found that C2-5 alkyl diesters of 1-6 exhibit excellent prodrug properties. Substantially higher levels of fluorinated nucleoside are observed in rat and monkey blood. In addition, the ratio of cytidine to uridine in the fluorinated base is higher in both species when the nucleoside was administered as the diester.

Additionally, diesters are capable of forming two different monoesters and the non-esterified nucleoside. Pharmacokinetic analysis in this situation can be complex if all species are present in blood at significant concentrations. The presence of multiple metabolites in the blood adds additional burdens to establish that the prodrug is safe. Surprisingly, hydrolysis of both esters is quite easy and the only significant metabolite observed in the blood other than the parent nucleoside is 31-monoester which is rapidly converted to 1-6.

To also evaluate potential behavior in humans, transport through Caco-2 cells was evaluated for the assumed prodrugs. Caco-2 cells are commonly used to assess the potential absorption / permeability of molecules (G. Gaviraghi et al. In Pharmacokinetic Optimization in Drug Research. Biological, Pharmacokinetic and Computational Strategies. B. Testa et al., Wiley Interscience VCHi Zurich 2001 pp 314). Caco-2 permeability is acceptable for C 2-6 alkyl diesters of 1-6.

In addition to efficient in vivo biotransformation, a prodrug for oral administration must also exhibit appropriate physicochemical properties to formulate the drug and ensure intestinal absorption in a compartment in which the desired biotransformation may occur. Particularly relevant are water solubility, division coefficient and stability in gastric and intestinal fluids. The values for these parameters are shown in Table 2. Table 2

<table> table see original document page 37 </column> </row> <table> 30

1 = Calculated octanol / auga split coefficient

2. Experimental distribution coefficient determined at pH 7,4

3. Solubility in aqueous medium, water, or pH 6.5 buffer (mg / mL).

4. Stability in simulated gastric fluid (SGF; pH 1.2)

5. Stability in simulated intestinal fluid (SIF; pH 7.4)

6. Solubility in SGF is 13.3 mg / mL

7. not determined

8. Medium is SIF, solubility in SGF is 13.6 mg / mL

9. Medium is SIF, solubility in SGF is 0.16 mg / mL

10. Measured in water and pH 6 buffer

11. Minor degradation noted over time

12. Determined at pH 5.5 The Po / w oil-water division coefficient (clogP is the calculated Po / w) is an important property for orally administered medicinal products. The drug substance must have sufficient aqueous solubility to dissolve in the formulation and gastrointestinal (GI) fluids to make contact with endothelial cells in the stomach and intestine and sufficient lipid solubility to pass through the lipid bilayer membrane of these cells and finally in the blood. The optimal range for P1 of an oral bioavailability compound is between 1 and 3 which is exhibited by compounds of the present invention.

The expression Po / w as used herein refers to the octanol / water division coefficient. The expression clogP as used herein refers to the calculated Po / w. Computer programs that calculate Po / w are easily available to the pharmaceutical and medicinal chemist. The term distribution coefficient refers to the experimentally determined coefficient of division between octanol and a buffered aqueous solution. The division coefficient and distribution coefficient are generally similar, but the latter is a function of the pH of the aqueous solution while the Po / w is independent of pH.

The solubility in water of a prodrug for oral administration should be greater than at least about 0.1 mg / mL for optimal formulation and the half life in simulated gastric fluid and simulated intestinal fluid should be 1-2 h. 2-4 h respectively to allow sufficient time to cross the stomach and be absorbed in the intestine.

The compounds of the present invention are conveniently prepared in one step by acylating 1-6 in an aqueous organic solvent. The solvent may be a homogeneous aqueous solution or a two phase solution. The pH of the aqueous organic solvent is maintained above 7.5 by addition of base to neutralize acid produced by acylation. The base may be an alkali or alkali metal hydroxide or a tertiary amine. The reaction is performed in the presence of DMAP which is known in the art to be a catalyst for acylation. An advantage of the present process is that the desired product can be obtained without acylation of the heterocyclic base. No protection groups are required which eliminates the need for a protection / unprotection step. The process is described in the examples.

Dosage and administration

The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms and carriers. Oral administration may be in the form of tablets, coated tablets, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions. Compounds of the present invention are effective when administered by suppository administration, among other routes of administration. The most convenient way of administration is generally oral with the use of a convenient daily dosage regimen that can be adjusted according to the severity of the disease and the patient's response to antiviral medication.

A compound or compounds of the present invention, as well as pharmaceutically useful salts thereof, together with one or more conventional excipients, carriers, or diluents, may be in the form of pharmaceutical and unit dosage compositions. Pharmaceutical compositions and unit dosage forms may comprise conventional ingredients in conventional proportions with or without additional active compounds and the unit dosage forms may contain any suitable effective amount of the active ingredient proportional to the intended daily dosage range to be employed. Pharmaceutical compositions may be employed as solids, such as filled tablets or capsules, semi-solids, powders, sustained release formulations, or liquids as suspensions, emulsions, or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration. A typical preparation will have from about 5% to about 95% active compound or compounds (w / w). The term "preparation" or "dosage form" should include solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingredient may exist in different preparations depending on the desired dose of pharmacokinetic parameters.

The term "excipient" as used herein refers to a compound that is used to prepare the pharmaceutical composition, and is generally safe, non-toxic and not biologically undesirable, and includes excipients that are acceptable for veterinary as well as human pharmaceutical use. The compounds of this invention may be administered alone but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with respect to the intended route of administration and standard pharmaceutical practice.

A "pharmaceutically acceptable salt" form of an active ingredient may also initially confer a desirable pharmacokinetic property on the active ingredient that was absent in unsalted form, and may be positively affected by the pharmacodynamics of the active ingredient with respect to its therapeutic activity in the body. The phrase "pharmaceutically acceptable salt" of a compound as used herein means a salt that is pharmaceutically acceptable and which has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and others; or formed with organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid. mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanesulfonic acid, 2-hydroxy ethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, lauryl sulfuric acid gluconic acid, glutamic acid, salicylic acid, muconic acid and others. It is to be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same acid addition salt.

Solid form preparations include dispersible powders, tablets, pills, capsules, suppositories, and granules. A solid carrier may be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is generally a finely divided solid which is a mixture with the finely divided active component. In tablets, the active ingredient is generally mixed with the carrier having the required binding capacity in appropriate proportions and compacted into the desired shape and size. Suitable carriers include, without limitation, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Solid form preparations may contain, in addition to the active ingredient, colorants, flavorings, buffer stabilizers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.

Liquid formulations are also suitable for oral administration and include aqueous emulsions, syrups, elixirs and suspensions. These include solid form preparations which must be converted to liquid form preparations just prior to use. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous suspensions may be prepared by dispersing the finely divided active component in water with viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

The compounds of the present invention may be formulated for administration as suppositories. A low melt wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is homogeneously dispersed, for example by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be suitable.

Suitable formulations together with pharmaceutical carriers, diluents and excipients are described in Remington: The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19th edition, Easton, Pennsylvania. A formulation skilled practitioner may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity.

Modification of the present compound to make it more soluble in water or another vehicle, for example, can easily be accomplished by minor modifications (e.g. salt formulation), which are within the ordinary practice in the art. It is also within common practice in the art to modify the route of administration and dosage regimen of a particular compound to control the pharmacokinetics of the present exposed for maximum beneficial effect in patients.

The term therapeutically effective holiness as used herein means an amount necessary to reduce the symptoms, the disease in an individual. The dose will be adjusted to the individual needs in each particular case. That numerous factors like the severity of the disease being treated, age, general health. patient's condition other medications with which the patient is being treated, the route and form of administration and the preferences and experience of the medical professional involved. For oral administration, a daily dosage of from about 0.1 to about 10 g per day should be suitable as monotherapy and / or combination therapy. A preferred daily dose is from about 0.5 to about 7.5 g per day, more preferably 1.5 to about 6.0 g per day. Generally, treatment is initiated with an initial large "loading dose" to rapidly reduce or eliminate the virus followed by a dose reduction to a level sufficient to prevent resurgence of infection. A person of ordinary skill in the treatment of the diseases described herein will be able, without undue experimentation and with confidence in personal knowledge, to experience the disclosures of this application, to determine the therapeutically effective amount of the compounds of the present invention for a given disease. patient.

Therapeutic efficacy can be determined from liver function tests that include, but are not limited to, protein levels such as serum proteins (eg, albumin, coagulation factors, alkaline phosphatase, aminotransferases (eg, alanine transaminase, aspartate transaminase), 51-nucleosidase, γ-glutaminyltranspeptidase, etc.), bilirubin synthesis, cholesterol, and bile acid synthesis; a hepatic metabolic function including, without limitation, carbohydrate metabolism, amino acid metabolism and ammonia.

Alternatively, therapeutic efficacy may be monitored by HCV-RNA measurement. The results of these tests will allow the dose to be optimized.

In embodiments of the invention, the active compound or a salt may be administered in combination with another antiviral agent such as ribavirin, another HCV nucleoside polymerase inhibitor, a non-nucleoside HCV polymerase inhibitor, an HCV protease inhibitor, a HCV helicase inhibitor or an HCV fusion inhibitor. When the active compound or its derivative or salt is administered in combination with another antiviral agent the activity may be increased in the parent compound. When treatment is combination therapy, such administration may be concomitant or sequential with respect to that of nucleoside derivatives. "Concurrent administration" as used herein includes administration of the agents at the same time or at different times. Administration of two or more agents at the same time may be accomplished by a single formulation containing two or more active ingredients or by substantially simultaneous administration of two or more dosage forms with a single active agent.

It should be understood that references therein to treatment extend to prophylaxis as well as to treatment of existing conditions. In addition, the term "treatment" of an HCV infection as used herein also includes treatment or prophylaxis of a disease or condition associated with or mediated by HCV infection, or the clinical symptoms thereof.

Example 1

(2R, 3R, 4R, 5R) -5- (4-amino-2-oxo-2H-pyrimidin-1-yl) -4-fluoro-4-methyl-3-propionyloxy-tetrahydro-furan-2-propionic acid ylmethyl ester (1-3)

<formula> formula see original document page 46 </formula>

To a suspension of 1-6 (30 g, 0.116 mol), DMAP (1.4 g, 11.57 mmol) in THF (300 mL) and water (150 mL) was added TEA (35.1 g, 0.347 mol). ) to obtain a clear solution (pH about 11). The reaction mixture was cooled to 5-10 ° C and propionic anhydride (30.1 g, 0.231 mol) was added dropwise to the stirred biphasic reaction. The pH was monitored and maintained at about 11-12 by simultaneous addition of KOH solution. The progress of the reaction was monitored by HPLC analysis and after propionyl chloride was added there was about 52% diester, 30% monoesters and 15% starting material. Additional propionic anhydride (45.2 g, 0.347 mol) was added dropwise under the conditions described above. The reaction mixture was allowed to stand overnight without stirring. HPLC of the organic phase indicated about 96% diester and about 2% monoester. The phases were separated and the aqueous phase extracted twice with THF (100 mL). The combined organic phases were washed with brine. The organic phase is filtered, evaporated and the residue dissolved in water (about 500 mL) and then diluted with IPA (about 100 mL). The resulting mixture is slowly cooled to room temperature with stirring. The resulting precipitate was filtered, washed with water and heptane, dried at about 600 ° C under vacuum for about 60 hours to give 3.45 g (80.3%) of 1-3 which was 98.75% pure by HPLC. Example 2

Isobutyric Acid (2R, 3R, 4R, 5R) -5- (4-amino-2-oxo-2H-pyrimidin-1-yl) -4-fluoro-3-isobutyryloxy-4-methyl-tetrahydro-furan-2-one ylmethyl ester (1-2)

To an ice-cold suspension of 1-6 (970 g, 3.74 mol) and DMAP (50 g, 0.412 mol) in THF (10 L) was added TEA (2.3 kg, 16.5 mol) and water (7 L) that produced a transparent solution. Isobutyryl chloride (3 equivalents) was slowly added to the stirred mixture maintaining the temperature at about 0 ° C. Further 1.2 then 0.7 equivalent of isobutyryl chloride was added until HPLC indicated that the reaction proceeded to completion (a total of about 1.95 kg). The reaction mixture was acidified with HCl to a pH of about 6.4 and the organic phase was washed with EtOAc (2 x 10 L). The combined extracts were washed with water (1 χ 15 L). The organic phase is filtered and concentrated in vacuo. The residue was dissolved in IPA (about 20 kg) and heptane (14.2 kg) was added. The solution was heated to about 74-75 ° C to yield a clear solution, then about 5 L was distilled off. The resulting solution was slowly cooled to room temperature. A precipitate formed at about 42-43 ° C. Cooling was continued slowly at 5 ° C then stirring overnight. The resulting solid was filtered and the filtrate was washed with IPA / heptane (1: 8) mixture (13.4 kg), dried under vacuum at about 60-70 ° C to yield 1.295 kg (86.65%) of 1-2 which was 99.4% pure by HPLC.

Example 3

(2R, 3R, 4R, 5R) -5- (4-amino-2-oxo-2H-pyrimidin-1-yl) -4-fluoro-2-isobutoxycarbonyloxymethyl-4-methyl-tetrahydro-furan-3- il isobutyl ester ester (1-4)

<formula> formula see original document page 48 </formula>

A suspension of 1-6 (700 mg), DMAP (33 mg) in THF (7 mL) was diluted with brine (7 mL). Diluted NaOH was added until the pH was about 11. The reaction mixture was cooled in an ice bath and isobutyl chloroformate (1.11 g) is added dropwise to the stirred biphasic reaction mixture keeping the pH at about 11 by adding NaOH as required. HPLC analysis indicated that most of the dicarbonate was contaminated with about 15% monocarbonates. 1 more eq. of isobutyl chloroformate was added dropwise to the ice solution. HPLC indicated almost complete conversion. The resulting mixture was allowed to stand overnight at room temperature. EtOAc (about 50 mL) was added and the pH of the aqueous phase adjusted to about 7.5 with HCl. The phases were separated and the organic phase was washed with water (3x) and evaporated to dryness to give a colorless solid (about 1.22 g). The solid is dissolved in hot acetone resulting in a clear solution that was slowly cooled to RT which produced a mass of solid. The solid was diluted with IPA (about 20 mL) which afforded a broth which was filtered then washed sequentially with IPA and then dried heptane to yield 0.85 g (68.5%) of 1-4 which was 97.5%. pure by HPLC.

Example 4

(2R, 3R, 4R, 5R) -5- (4-amino-2-oxo-2H-pyrimidin-1-yl) -4-fluoro-4-methyl2-propoxycarbonyloxymethyl-tetrahydro-furan-3-yl ester propyl ester; hydrochloride salt (1-5)

To a suspension of 1-6 (700 mg, 2.70 mmol), DMAP (33 mg, 0.27 mmol) in THF (7 mL) and dilute brine (7 mL) was added diluted KOH to adjust the pH to about The reaction was cooled to about 5 ° C and n-propyl chloroformate (1.0 g) was added dropwise to the stirred biphasic reaction mixture. HPLC analysis indicated the formation of the desired product together with about 20% monocarbonates. Two additional amounts of propyl chloroformate (2x1 equivalent) were added to the cold solution until HPLC analysis indicated that the reaction proceeded to completion. The reaction mixture was diluted with EtOAc (30 mL) and the pH of the aqueous phase adjusted to about 6.5 with HCl. The phases were separated and the organic phase was washed with water (3x) and evaporated to dryness to obtain a colorless solid (about 1.1 g). A hot IPA solution was acidified with 4N HCl (about 1 mL) and evaporated to dryness. The resulting solid was redissolved in hot EtOH (about 115 mL) and stirred overnight at RT. A solid mass is formed which was filtered and the residue washed with MeOH / heptane (1: 1). The remaining solid was dried in vacuo at about 60 ° C to yield 0.325 g (25.8%) of 1-5 which was 97.5% pure by HPLC assay.

Example 5

Determination of pharmacokinetic parameters in rats Intact male mice XGS Wistar Han

Crl: WI (GLx / BRl / Han) IGS BR (Hanover-Wistar) weighing 200-250 g were used. Groups of three rats were used for each dose level of an experimental compound. The animals had normal access to feed and water throughout the experiment. The test substance was formulated as an aqueous suspension containing Captex355EP, Capmul MCM, EtOH, and propylene glycol (30: 20: 20: 30) at a dose equivalent to 10 mg / kg of 1-6 and was administered orally by forced feeding. A blood sample (0.3 mL) was collected from treated rats at 0.25, 0.5, 1, 3, 5, and 8 h from a jugular cannula and at 24 h by cardiac puncture. Potassium oxalate / NaF were added to the samples which were stored on ice during the sample procedure.

Samples were centrifuged in a -4 ° C refrigerated centrifuge as soon as possible and plasma samples were stored in a -800 ° C freezer until analysis. Aliquots of plasma (0.05 mL) were mixed with 0.1 mL of acetonitrile. Internal standard (0.05 mL in water) and 0.02 mL solvent were added. A set of calibration standards was prepared by mixing 0.05-mL aliquots of untreated rat plasma with 0.1 mL acetonitrile, 0.02-mL aliquots of standard solution in methanol: water (1: 1). and 0.05-mL aliquots of the internal standard in water. Each plasma sample and calibration standard was completely swirled and then centrifuged at 3,000 rpm for 5 minutes to precipitate the protein. Supernatant (100 μL each) from the centrifugation was transferred to a 96-well plate containing 200 μL aqueous mobile phase for LC / MS / MS analysis.

Sample Analysis:

Prodrugs were analyzed using high performance liquid chromatography with tandem mass spectrometry (HPLC / MS / MS). A Thermo Aquasil C18 4.6 χ 50 mm (5 μΜ) column was used for separation. Electrospray ionization (ESI) was used for the ionization process. Mobile phase A contained 5 mM ammonium acetate in water with 0.1% formic acid and mobile phase B contained MeOH with 0.1% formic acid. Elution was performed with the following gradient with a flow rate of 1 ml / min .:

<table> table see original document page 51 </column> </row> <table> <table> table see original document page 52 </column> </row> <table>

Example 6

Determination of pharmacokinetic parameters in monkeys

Three male Cynomolgus monkeys weighing 8-10 kg were used. The animals had normal access to feed and water throughout the experiment. Animal weights at the time of drug administration and unwanted animal reactions were recorded. The test substance was formulated as an aqueous suspension containing hypromellose (2910, 50 cps), USP, polysorbate 80, NF, benzyl alcohol, NF (5.0, 4.0 and 9.0 mg / mL) and sterile water ( sufficient amount to generate 1.0 mL) at a dose equivalent to 10 mg / kg of 1-6 and 0.5 mL / kg was administered orally by forced feeding. A blood sample (0.5 - 1.0 mL) was collected at 0, 0.083, 0.25, 0.5, 1, 3, 5, 8 and 24 hours. Sample handling and analysis was performed as described in the rat experiment. A 5 mL urine sample was taken from each monkey prior to dosing at 0-8 h.

Urine samples were stored at -80 ° C and analyzed by LC / MS / MS. Standard curves were prepared in empty monkey plasma containing NaF and potassium oxalate.

Example 7

Shard Protocol

Materials:

Krebs-Henseleit buffer powders, calcium chloride hydrate, and sodium bicarbonate were purchased from Sigma (St. Louis, MO). Caco-2 cells (Passage -100) were obtained from Roche Basel. DMEM / Upper Medium, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), and bovine serum were obtained from JRH Bioscience (Lenexa, KS). MEM Nonessential amino acids, L-glutamine, penicillin, and streptomycin were obtained from GIBCO Labs, Life Tech. LLC (Grand Island, NY). Snapwell cell culture inserts (6.5 mm in diameter, 1.12 cm2, 0.4 μπι pore size) were obtained from Costar (Cambridge, MA).

Cell cultures:

Cells were grown in 75-cm2 flasks and maintained at 370 ° C in an atmosphere of 5% CO2 and 95% air. The culture medium consisted of DMEM / upper medium supplemented with 5% bovine serum, 25 mM HEPES, 1% MEM nonessential amino acids, 1% L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cultures were passed each week in a division ratio of 1-3. For permeability studies, Caco-2 cells at pass number 110-120 were plated at a density of 400,000 cells / cm2 on Transwell polycarbonate filters in Snapwell inserts and left in culture for 7 days prior to use.

Krebs-Henseleit Cap:

Krebs-Henseleit bicarbonate buffer containing 10 mM glucose and 2.5 mM CaCl2 adjusted to pH 6.5 and 7.4 were prepared per package instructions. Powdered salts were quantitatively dissolved in about 90% of the required volume with Millipore water. Calcium chloride dihydrate and sodium bicarbonate were added prior to pH adjustment with IN HCl or IN NaOH. Additional Millipore water was added to bring the solution to a final volume. The solution was sterile filtered using a 0.22 micron porosity membrane and stored in the refrigerator (-20 ° C) until use.

Cell Preparation:

Differentiated cells were obtained from Cell Culture Core Facility and allowed to equilibrate at 37 ° C in an atmosphere of 5% CO2 and 95% air. Snapwells inserts containing caco-2 monolayers were rinsed in Krebs-Henseleit buffer equilibrated at 37 ° C pH 7.4. Method:

Cell insertion was used as the diffusion chamber. The pH of the Krebs-Henseleit buffer in the apical and basolateral chambers was 6.5 and 7.4, respectively, and the initial substrate concentration on the apical side was 100 μΜ. The cells with compound tested in the apical chamber were preincubated for approximately 30 minutes at 370 ° C under an atmosphere of 5% CO 2 and 95% air. The experiments were initiated when the 100 μΜ compound cell inserts in Krebs-Henseleit buffer at pH 6.5 were transferred to a new basolateral chamber pre-equilibrated buffer plate. Donor side samples at 0 minutes, donor and recipient sides at 30 minutes were collected for analysis. Post-Experiment Control:

Yellow Lucifer was used to evaluate the performance of the diffusion system. After the last sampling for the test compounds, Lucifer yellow was added to the apical chamber to generate an initial concentration of 100 μΜ. After 60 minutes of incubation, 250 μL were removed from the basal chamber and analyzed.

Calculation of permeability coefficient (Papp): Papp was calculated using the following formula:

<formula> formula see original document page 55 </formula>

When V is the volume (cm3) of the receptor solution, A p is the surface area (cm2) of the Snapwell insert, Co is the initial concentration (nM), and dC / dt is the change in concentration in the receiving chamber over time, that is, the slope (nM / min) of concentration in the receiving chamber vs. time. Concentrations at each time point in the sample were corrected to account for aliquots removed or transferred from donor inserts to new plates depending on the experiment. Example 8

Pharmaceutical compositions of the present compounds for administration via various routes were prepared as described in Example 8.

Composition for wet granular formulation for oral administration (A):

<table> table see original document page 55 </column> </row> <table> <table> table see original document page 56 </column> </row> <table>

The ingredients are mixed, granulated and filled into hard gelatin capsules containing about 500 mg of active compound.

Composition for oral administration (B):

<table> table see original document page 56 </column> </row> <table>

The ingredients are combined and granulated using a solvent such as water. The formulation is then dried and tableted containing about 500 mg of active compound with a suitable tabletting machine.

Composition for oral administration (C)

<table> table see original document page 56 </column> </row> <table>

The ingredients are mixed to form a suspension for oral administration. Suppository Formulation (D):

<table> table see original document page 57 </column> </row> <table>

The ingredients are melted and mixed on a steam bath and poured into molds containing 2.5 g total weight.

The characteristics disclosed in the foregoing description, or the following claims, expressed either in their specific forms or in terms of a means for performing the disclosed function, or a method or process for achieving the disclosed result, as appropriate, may separately or in any combination of such features may be used for carrying out the invention in various forms thereof.

The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. It will be obvious to one skilled in the art that changes and modifications may be made within the scope of the appended claims. Therefore, it should be understood that the above description should be illustrative and not restrictive. The scope of the invention should therefore be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, together with the full scope of equivalents to which such claims are designated.

All patents, patent applications and publications cited in that application are hereby incorporated in their entirety by reference for all purposes to the same extent as if each individual patent, patent application or publication were individually indicated.

Claims (17)

1. A compound of formula I <formula> formula see original document page 59 </formula> characterized in that: R1 is selected from the group consisting of branched or unbranched C2-5 alkyl, branched or unbranched C2-5 alkenyl C 2-5 branched or unbranched alkynyl, C 2-5 lower haloalkyl, C 3-6 cycloalkyl, and C 2-4 alkoxy; and hydrates, solvates and acid addition salts thereof. A compound according to claim 1, characterized in that R 1 is ethyl, n-propyl, iso-drODyl n-butyl-nri-hnt- A compound according to claim 1 or 2, characterized in that R 1 is ethyl or isopropyl. Compound according to Claims 1 to 3, characterized in that R 1 is isopropyl and the compound is the hydrochloride or sulfate salt. Compound according to Claims 1 to 4, characterized in that R1 is isopropyl and the compound is the hydrochloride salt. A compound according to claim 1, characterized in that R 1 is ethoxy, n-propyloxy or isopropyloxy. Compound according to Claims 1 to 6, characterized for use in therapy. A compound according to claims 1 to 6, characterized for use in the therapy of a hepatitis C virus (HCV) mediated disease. Pharmaceutical composition comprising a therapeutically effective amount of a compound according to claims 1 to 6 mixed with at least one pharmaceutically acceptable carrier, diluent or excipient. Use of a compound according to claims 1 to 6 for the preparation of a medicament for the treatment of a hepatitis C virus (HCV) mediated disease. Use according to claim 10, characterized in that the medicament is administered to a patient in need thereof in a therapeutically effective dose. Use according to claim 10 or 11, characterized in that the medicament is administered to a patient in need thereof at a dose of between 0.1 and 10 g per day. Use according to claims 10 to 12, characterized in that the medicament is also administered with at least one immune system modulator and / or at least one antiviral agent that inhibits HCV replication. A method for treating a hepatitis C virus (HCV) mediated disease comprising administering to a patient in need thereof a therapeutically effective dose of a compound according to claims 1 to 6. The method of claim 14, characterized in that the dose between 0.1 and 10 g per day is administered to the patient. The method of claims 14 or 15, further comprising administering at least one immune system modulator and / or at least one antiviral agent that inhibits HCV replication. 17. A characterized process for selective O-acylation of a nucleoside I to generate an O-acyl nucleoside II under basic reaction conditions wherein R1 is selected from the group consisting of C2-5 branched or unbranched alkyl, C2-5 branched or unbranched alkenyl, C2-5 branched or unbranched alkynyl, C2-5 lower haloalkyl, C3-6 cycloalkyl, and C2-4 alkoxy whose process comprises the steps of: (i) dissolving II and DMAP in a mixture of aqueous heterogeneous solvent and adding aqueous base to adjust the pH from about 7.5 to about 12; (ii) optionally adding sufficient saturated aqueous NaCl to produce a biphasic reaction mixture; (iii) adding an acylating agent and additional base sufficient to maintain a pH of from about 7.5 to about 12; (iv) monitoring the reaction and discontinuing the addition of said acylating agent and said base when the conversion reaches a satisfactory level; (ν) optionally contacting O-acyl nucleoside a pharmaceutically acceptable acid to form an acid addition salt of the O-acyl nucleoside.